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ItemDeciphering the signalling pathways associated with neuronal death in excitotoxicity by quantitative proteomic approachesAmeen, Syeda Sadia ( 2019)Excitotoxicity is a primary pathological process directing neuronal cell death in both acute neurological disorders and neurodegenerative diseases such as ischemic stroke and Alzheimer’s disease. It is initiated by over-stimulation of ionotropic glutamate receptors (iGluRs), which in turn permit the influx of excessive calcium ions into the cytosol of the affected neurons. The sustained and excessively high cytosolic calcium concentration induces substantial imbalance of the intracellular environment such as depletion of ATP and over-production of reactive oxygen species by inducing dysregulation of specific proteases, protein kinases, and phosphatases. These dysregulated enzymes in turn aberrantly catalyse post-translational modifications of specific neuronal proteins to cause neuronal death. Despite the efforts by many investigators in the past four decades, the cell signalling pathways relaying the neurotoxic signals from the over-stimulated iGluRs and the excessively high cytosolic calcium concentration to direct neuronal death in excitotoxicity, remains unclear. Avenues to chart these neurotoxic signalling pathways include identifying the excitotoxicity-dysregulated proteases, protein kinases, and phosphatases, and delineating the mechanisms of their dysregulation and elucidating their exact roles in excitotoxic neuronal death. In my PhD study, I employed multiple quantitative proteomic approaches and a combination of the neuronal cell-based model and two animal models of excitotoxicity to identify the neuronal proteins aberrantly modified by these excitotoxicity-dysregulated enzymes. Furthermore, these approaches also defined the exact sites of modifications and quantified the degree of modifications. The proteomic approaches I adopted include the quantitative N-terminomic, global and phosphoproteomic approaches. They were used to analyse changes in general protein degradation, limited proteolysis, phosphorylation and dephosphorylation of specific neuronal proteins catalysed by the excitotoxicity dysregulated enzymes in cultured primary cortical neurons and in brain tissues subjected to ischemic stroke and traumatic brain injury. Using an N-terminomics method called “Terminal amine isotopic labelling of substrates” (TAILS), I discovered hundreds of cellular proteins undergoing enhanced proteolytic processing catalysed by the excitotoxicity-activated proteases to form truncated protein fragments in neurons over-stimulated with glutamate. To define the identities of these excitotoxicity-associated proteases, I performed TAILS analysis of neurons co-treated with the excitotoxic level of glutamate and calpeptin, a specific inhibitor of calpains and cathepsins. Results of my analysis revealed that calpeptin abolished enhanced proteolytic processing of the majority of the neuronal proteins identified in the TAILS study of neurons treated with glutamate-only. These findings indicate that most of these identified neuronal proteins were either direct calpain/cathepsin substrates or the substrates of proteases activated by calpains or cathepsins. More importantly, the findings suggest that calpains and cathepsins are the major modulator proteases catalysing proteolytic processing of specific neuronal proteins in excitotoxicity. Using label-free global and phosphoproteomics analyses I found 483 phosphopeptides derived from neuronal proteins undergoing significant changes in phosphorylation level in glutamate-treated primary cortical neurons. Interestingly, global proteomic analysis revealed only a few neuronal proteins exhibiting significant changes in abundance, suggesting that most of the neuronal proteins remained stable up to 240 min of glutamate treatment. These findings suggest that instead of protein synthesis and degradation, phosphorylation plays a major role in modulating the activities and functions of specific neuronal proteins in excitotoxicity. From the identified phosphosites in the neuronal proteins undergoing significant changes in phosphorylation state, bioinformatic analysis predicted PAK1 (Serine/threonine-protein kinase), CK2A1 (Casein kinase II subunit alpha), and mTOR (mammalian target of rapamycin) as the most perturbed kinases in neurons undergoing excitotoxic cell death. Bioinformatic analysis of the neuronal proteins showing significantly changed phosphorylation and/or enhanced proteolytic processing predicted defective axonal guidance signalling as the most perturbed signalling pathway in excitotoxicity. To complement the proteomic analysis in cultured neurons, I conducted phosphoproteomic studies of thebrains in mice suffering from stroke and TBI (traumatic brain injury). Results of my study indicate that neuronal proteins, especially those in synapses are mostly dysregulated. Brain tissue lysates contain proteins expressed in neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells. The high abundance of neuronal proteins identified as the brain proteins exhibiting significant changes in phosphorylation state induced by stroke and TBI suggest that neurons are particularly sensitive to the detrimental impacts of stroke and TBI. The neuronal protein tyrosine kinase Src was discovered by TAILS to be cleaved by calpains to form a truncated Src fragment in excitotoxicity. To examine the therapeutic potential of my proteomic findings, I chose to investigate if blockade of calpain cleavage of Src in neurons could protect against neuronal loss in vivo in a rat model of neurotoxicity. My study showed that stereotaxic injection of a cell membrane-permeable peptide inhibitor of calpain cleavage of Src in rats can protect against neuronal loss induced by over-stimulation of the N-methyl-D-aspartate (NMDA) receptors. Taken together, results of my proteomic analyses have built a conceptual framework for future investigation to decipher the signalling pathways underpinning neuronal death in excitotoxicity. Furthermore, my results provided evidence of the therapeutic potential of the findings from TAILS analysis. Some of the neuronal proteins identified in my study to exhibit significantly perturbed phosphorylation state and/or proteolytic processing, are potential targets for the development of new therapeutic strategies to reduce neuronal loss in ischemic stroke, traumatic brain injury and other neurodegenerative diseases.